The influence of an endogenous β3 subunit on recombinant GABA A receptor assembly and pharmacology in WSS-1 cells and transiently transfected HEK293 cells

Abstract

Cell lines are commonly used for studying recombinant heterooligomeric ion channels with defined subunit composition. Such studies often ignore the contribution of endogenous proteins in the assembly of mature channels. We examined whether an endogenous subunit was required for the functional expression of γ-aminobutyric acid type A (GABA A) receptors in WSS-1 cells, HEK293 cells stably expressing recombinant α1 and γ2 subunits. Our pharmacological and RT-PCR analyses of GABA A receptors and their mRNAs in WSS-1 cells confirm the presence of α1 and γ2 subunits and suggest the existence of an endogenous β3 subunit. Whole-cell GABA-evoked currents recorded from untransfected WSS-1 cells were blocked by bicuculline methiodide and enhanced by anesthetics and anticonvulsants including the subunit-selective compounds diazepam and loreclezole. These data suggest that, in addition to the γ2 subunit, WSS-1 cell receptors also contain β2/3 subunits. RT-PCR revealed that WSS-1 cells and parental HEK293 cells contain β3 mRNA. We examined the contribution of the β3 subunit in the function of receptors formed by expression of α1 and γ2S subunits. Untransfected HEK293 cells were unresponsive to GABA. Cells transfected with α1 and γ2S cDNAs displayed small diazepam and loreclezole responsive GABA-activated currents. By contrast, the expression of α1 and γ2S cDNAs in the neuroblastoma NB41A3 cell line, that lacks β subunit mRNAs, failed to produce functional receptors. These data reaffirm that α1 and γ2S subunits alone do not form functional GABA A receptors and that receptors of WSS-1 cells contain α1, β3 and γ2S subunits.